Fucosidase from bacteroides and methods using the same

ABSTRACT

The present disclosure relates to an α-fucosidase having α-(1,2), α-(1,3), α-(1,4), and α-(1,6) fucosidase activity. The present disclosure also relates to the compositions comprising the α-fucosidase, and the methods of producing and using the α-fucosidase in cleaving α-(1,2), α-(1,3), α-(1,4), and/or α-(1,6)-linked fucoses in the glycoconjugates.

RELATED APPLICATIONS

This application claims the benefit of US provisional applications U.S.Ser. No. 62/003,136 filed May 27, 2014, U.S. Ser. No. 62/003,104 filedMay 27, 2014, U.S. Ser. No. 62/003,908 filed May 28, 2014, U.S. Ser. No.62/020,199 filed Jul. 2, 2014 and U.S. Ser. No. 62/110,338 filed Jan.30, 2015. The contents of which is hereby incorporated in its entirety.

BACKGROUND OF THE INVENTION

Fucose is an important component of many O- or N-linked oligosaccharidestructures of glycoconjugates. Fucose-containing glycans are involved innumerous biological events, including development and apoptosis, and areinvolved in the pathology of inflammation, cancer, and cystic fibrosis.Defucosylation of the glycoconjugates is an important process forunderstanding the biological effects of the glycoconjugates.

α-L-fucosidases (α-fucosidase) are exo-glycosidases, responsible for theremoval of fucose residues from the non-reducing end of glycoconjugatesby hydrolyzing α-(1,2), α-(1,3), α-(1,4), and α-(1,6) linkages offucoses attached, primarily to galactose or N-acetylglucosamine.

Both human serum IgG and therapeutic antibodies are well known to beheavily fucosylated. Antibody-dependent cellular cytotoxicity (ADCC) hasbeen found to be one of the important effector functions responsible forthe clinical efficacy of therapeutic antibodies. ADCC is triggered uponthe binding of lymphocyte receptors (FccRs) to the antibody Fc region.ADCC activity is dependent on the amount of fucose attached to theinnermost GlcNAc of N-linked Fc oligosaccharide via an α-(1,6) linkage.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides the compositions and methodsfor the improved enzymatic hydrolysis of fucose in vitro. In particular,the present invention is useful for the efficient cleavage of corefucose in native glycoproteins without denaturation or functionaldeterioration of glycoproteins. The compositions and methods of theinvention can facilitate the Fc glycoengineering of Fc fusion proteinsor antibodies, such as therapeutic antibodies. This invention alsoprovides the application of glycan sequencing for distinguishing fucoseposition on a glycoconjugate. The glycoconjugate may be a glycolipid,glycoprotein, oligosaccharide, or glycopeptide.

In one aspect, the present invention relates to an α-fucosidasecomprising a polypeptide having at least 85% sequence identity to SEQ IDNO: 1. In some embodiments, the α-fucosidase comprises a polypeptidehaving at least 88% sequence identity to SEQ ID NO: 1. In someembodiments, the α-fucosidase comprises a polypeptide having thesequence identity to SEQ ID NO: 1. In certain embodiments, theα-fucosidase comprises a polypeptide having the sequence identity to SEQID NO: 2. SEQ ID NOs: 1 and 2 share 88% sequence identity.

The fucosidase described herein can hydrolyze one or more α(1,2),α(1,3), α(1,4), and α(1,6)-linked fucoses. The fucoses may be present inN- and/or O-linked glycans in a glycoconjugate. In certain embodiments,the α-fucosidase is a recombinant Bacteroides α-fucosidase.

In preferred embodiments, the α-fucosidase exhibits pH optimum at 4-9.

In another aspect, the present invention relates to a compositioncomprises the α-fucosidase described above. The composition may furthercomprise at least one glycosidase. In some embodiments, the glycosidasemay be an exoglycosidase. The exoglycosidase includes, but not limitedto, sialidase, galactosidase, alpha-fucosidase, and variants thereof. Insome embodiments, the glycosidase may be an endoglycosidase. Theendoglycosidase includes, but not limited to,Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3,EndoH, EndoM, EndoS, and variants thereof.

The composition of the invention is useful for making defucosylation ofa glycoconjugate in vitro. In particular, the composition describedherein is useful for making core defucosylation of glycoproteins invitro. In some embodiments, the core defucosylation is core α(1,6)defucosylation. In certain embodiments, the core defucosylation is coreα(1,3) defucosylation. The defucosylation can be performed withoutdenaturation or functional deterioration of glycoproteins.

Another aspect of the invention provides a method for makingdefucosylation of a glycoconjugate in vitro. The inventive methodcomprises the step of contacting the glycoconjugate with theα-fucosidase of the invention described above. The glycoconjugatecomprises one or more fucoses selected from α(1,2), α(1,3), α(1,4), andα(1,6)-linked fucoses. The fucoses may be present in N- and/or O-linkedglycans in a glycoconjugate.

In some embodiments, the glycoconjugate is a glycoprotein. In someembodiments, the glycoprotein comprises a core fucose. In someembodiments, the core fucose is a core α-(1,3)-linked fucose or a coreα-(1,6)-linked fucose.

In some embodiments, the method further comprises contacting theglycoconjugate with at least one glycosidase. In certain embodiments,the glycosidase is an endoglycosidase. Endoglycosidase is used to trimoff the variable portions of an oligosaccharide in the N-glycan.Examples of endoglycosidases used herein include, but not limited to,Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3,EndoH, EndoM, EndoS, and variants thereof. Exoglycosidase

For core defucosylation, the glycoconjugate can be treated with anendoglycosidase and an α-fucosidase sequentially or simultaneously. Thecore defucosylation may be core α(1,3) defucosylation or α(1,6)defucosylation.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. shows the biochemical properties of BfFucH.

FIG. 2. (a) pH profile of BfFucH (b) temperature effects on the enzymeactivity of BfFucH (c) metal ion effects on the enzyme activity ofBfFucH.

FIG. 3. shows the time course of BfFucH treatment for Rituxan.

DETAILED DESCRIPTION OF THE INVENTION

The absence of core fucose residues in the Fc glycans is known tosubstantially increase the ADCC activity of IgG as nonfucosylatedantibodies bind to the FcgRIIIα receptor with significantly increasedaffinity. To improve FcgRIIIα binding and ADCC, several strategies havebeen developed to reduce fucosylation of IgG, including the developmentof production cell lines that abolish or reduce expression levels ofα-(1,6) fucosyltransferase. Alternative strategies to reducefucosylation include silencing the α-(1,6) fucosyltransferase gene usingRNAi. However, core defucosylation of N-glycans has not been able to beachieved enzymatically in vitro, mainly because N-glycans are embeddedbetween two Fc domains. The enzymatic defucosylation efficiency is muchlower due to steric hindrance, i.e., access of α-fucosidase to fucoseresidues is blocked by portions of the Fc domains.

A number of α-fucosidases are known in the art. Examples includeα-fucosidases from Turbo cornutus, Charonia lampas, Bacillus fulminans,Aspergillus niger, Clostridium perfringens, Bovine kidney (Glyko),Chicken liver (Tyagarajan et al., 1996, Glycobiology 6:83-93) andα-fucosidase II from Xanthomonas manihotis (Glyko, PROzyme). Somefucosidase are also commercially available (Glyko, Novato, Calif.;PROzyme, San Leandro, Calif.; Calbiochem-Novabiochem Corp., San Diego,Calif.; among others). None of these α-fucosidases are able toefficiently cleave the core fucoses from N-linked glycans withoutdenaturing the glycoproteins first.

WO 2013/12066 disclosed the defucosylation of (Fucαl,6) GlcNAc-Rituximabby an α-fucosidase from bovine kidney. As described in WO 2013/12066, areaction mixture of (Fuc αl, 6) GlcNAc-Rituximab was incubated withα-fucosidase from bovine kidney (commercially available from Prozyme) at37° C. for 20 days to completely remove the fucose in (Fucαl,6)GlcNAc-Rituximab. Thermal instability of immunoglobulin is known in theart (Vermeer et al., Biophys J. Jan 78: 394-404 (2000)). The Fabfragment is most sensitive to heat treatment, whereas the Fc fragment ismost sensitive to decreasing pH. It is contemplated that the antibodywill significantly lose the binding affinity to CD20 after prolongedthermal treatment, such as at 37° C. for 20 days, as described in WO2013/12066.

The limitation of currently known α-fucosidases has prevented effectivemanipulation of certain N-linked glycans. Thus, a need remains for newα-fucosidases suitable for Fc glycoengineering of Fc fusion proteins orantibodies for development of human therapeutics.

The present disclosure relates to an unexpected discovery of a bacterialα-fucosidase that is able to efficiently cleave core fucose fromN-linked glycans.

The present disclosure relates to an unexpected discovery of a bacterialα-fucosidase that is able to efficiently cleave core fucoses fromN-linked glycans.

In some examples, the α-fucosidase may be an α-fucosidase fromBacteroides fragilis (BfFucH). In some examples, the α-fucosidase may bean α-fucosidase from Bacteroides thetaiotaomicron (BtFucH). Theα-fucosidase can be expressed from bacteria, yeast, baculovirus/insect,or mammalian cells. In some embodiments, the α-fucosidase can be arecombinant Bacteroides α-fucosidase. In some embodiments, theα-fucosidase can be a recombinant Bacteroides α-fucosidase expressedfrom E. coli.

The α-fucosidase can hydrolyze one or more α(1,2), α(1,3), α(1,4), andα(1,6)-linked fucoses. The fucose may be present in N- and/or O-linkedglycans in a glycoconjugate. The fucose can be a core α-(1,3) fucose ora core α-(1,6) fucose.

Scheme 1 shows various fucose-containing glycoconjugates.

Examples of the substrates suitable for the enzyme include, but notlimited to, milk oligosaccharides, cancer associated carbohydrateantigens such as Globo H, Lewis blood groups (a, b, x, y), and sialylLewis a (SLe^(a)) and x (SLe^(x)). Unlike the reports known in the arts,the α-fucosidase can hydrolyze sialyl Lewis a (SLe^(a)) and x (SLe^(x))without cleaving the terminal sialic acid. Milk oligosaccharides maybear α-(1,2), α-(1,3) and/or α-(1,4) linked fucoses.

Compositions

The present invention also relates to a composition of the α-fucosidasedescribed above. The α-fucosidase comprises a polypeptide having atleast 85% sequence identity to SEQ ID NO: 1. In some embodiments, theα-fucosidase comprises a polypeptide having at least 88% identity to SEQID NO: 1, or a functional variant thereof. In some embodiments, theα-fucosidase comprises a polypeptide having an amino acid sequence ofSEQ ID NO: 1. In some embodiments, the α-fucosidase comprises apolypeptide having an amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2has 88% sequence identity to SEQ ID NO: 1.

Variant polypeptide as described herein are those for which the aminoacid sequence varies from that in SEQ ID NO: 1 or 2, but exhibit thesame or similar function of the enzyme comprising the polypeptide havingan amino acid sequence of SEQ ID NO: 1 or 2.

TABLE 1 SEQ ID: 1QQKYQPTEANLKARSEFQDNKFGIFLHWGLYAMLATGEWTMTNNNLNYKEYAKLAGGFYPSKFDADKWVAAIKASGAKYICFTTRHHEGFSMFDTKYSDYNIVKATPFKRDVVKELADACAKHGIKLHFYYSHIDWYREDAPQGRTGRRTGRPNPKGDWKSYYQFMNNQLTELLTNYGPIGAIWFDGWWDQDINPDFDWELPEQYALIHRLQPACLVGNNHHQTPFAGEDIQIFERDLPGENTAGLSGQSVSHLPLETCETMNGMWGYKITDQNYKSTKTLIHYLVKAAGKDANLLMNIGPQPDGELPEVAVQRLKEVGEWMSKYGETIYGTRGGLVAPHDWGVTTQKGNKLYVHILNLQDKALFLPIVDKKVKKAVVFADKTPVRFTKNKEGIVLELAKVPTDVDYVVELTID SEQ ID: 2QSSYQPGEENLKAREEFQDNKFGIFLHWGLYAMLATGEWTMTNNNLNYKEYAKLAGGFYPSKFDADKWVAAIKASGAKYICLTSRHHDGFSMFDTQYSDFNIVKATPFKRDIIKELAAACSKQGIKLHFYYSHLDWTREDYPWGRTGRGTGRSNPQGDWKSYYQFMNNQLTELLTNYGPVGAIWFDGWWDQDGNPGFNWELPEQYAMIHKLQPGCLIGNNHHQTPFAGEDIQIFERDLPGENTAGLSGQSVSHLPLETCETMNGMWGYKITDQNYKSTKTLIHYLVKAAGKNANLLMNIGPQPDGELPEVAVQRLKEMGEWMNQYGETIYGTRGGAVAPHDWGVTTQKGNKLYVHILNLQDKALFLPLADKKVKKAVLFKNGTPVRFTKNKEGVLLEFTEIPKDIDYVVELTID

As used herein percent (%) sequence identity with respect to a sequenceis defined as the percentage of amino acid residues in a candidatepolypeptide sequence that are identical with the amino acid residues inthe reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity. Alignment for purposes of determining percent sequenceidentity can be achieved in various ways that are within the skill inthe art, for instance, using publicly available computer software suchas BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

It will be understood that the polypeptide of the α-fucosidase of theinvention may be derivatized or modified to assist with their isolationor purification. Thus, in one embodiment of the invention, thepolypeptide for use in the invention is derivatized or modified byaddition of a ligand which is capable of binding directly andspecifically to a separation means. Alternatively, the polypeptide isderivatized or modified by addition of one member of a binding pair andthe separation means comprises a reagent that is derivatized or modifiedby addition of the other member of a binding pair. Any suitable bindingpair can be used. In a preferred embodiment where the polypeptide foruse in the invention is derivatized or modified by addition of onemember of a binding pair, the polypeptide is preferably histidine-taggedor biotin-tagged. Typically the amino acid coding sequence of thehistidine or biotin tag is included at the gene level and the proteinsare expressed recombinantly in E. coli. The histidine or biotin tag istypically present at one end of the polypeptide, either at theN-terminus or at the C-terminus. The histidine tag typically consists ofsix histidine residues (SEQ ID NO: 19), although it can be longer thanthis, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, forexample 5, 4, 3, 2 or 1 amino acids. Furthermore, the histidine tag maycontain one or more amino acid substitutions, preferably conservativesubstitutions as defined above.

Applications of the Compositions

The composition of the invention can be used for making defucosylationof a glycoconjugate in vitro. The inventive method comprises the step ofcontacting the glycoconjugate with the α-fucosidase of the inventiondescribed above. The glycoconjugate comprises one or more fucosesselected from α-(1,2), α-(1,3), α-(1,4), and α-(1,6)-linked fucoses. Thefucoses may be present in N- and/or O-linked glycans in aglycoconjugate.

In some embodiments, the glycoconjugate is a glycoprotein. In someembodiments, the glycoprotein comprises a core fucose. In someembodiments, the core fucose is a core α-(1,3) linked fucose or a coreα-(1,6) linked fucose.

In some embodiments, the method further comprises contacting theglycoconjugate with at least one glycosidase. In certain embodiments,the glycosidase is an endoglycosidase. Endoglycosidase is used to trimoff the variable portions of an oligosaccharide in the N-glycan.Examples of endoglycosidases used herein include, but not limited to,Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3,EndoH, EndoM, EndoS, and variants thereof.

For core defucosylation, the glycoconjugate can be treated with anendoglycosidase and an α-fucosidase sequentially or simultaneously. Thecore defucosylation may be core α(1,3) defucosylation or α(1,6)defucosylation.

The method of the invention can be useful for making Fc glycoengineeringfrom monoclonal antibodies. Exemplary methods of engineering aredescribed in, for example, Wong et at U.S. Ser. No. 12/959,351, thecontents of which is hereby incorporated by reference. Preferrably, themonoclonal antibodies are therapeutic monoclonal antibodies. In someexamples, the method for making a homogeneously glycosylated monoclonalantibody comprises the steps of (a) contacting a monoclonal antibodywith an α-fucosidase and at least one endoglycosidase, thereby yieldinga defucosylated antibody having a single N-acetylglucosamine (GlcNAc),and (b) adding a carbohydrate moiety to GlcNAc under suitableconditions. In certain embodiments, the glycan can be prepared bytreatment with endo-GlcNACase and exemplary fucosidase, then followed byexemplary endo-S mutant and a glycan oxazoline.

In a specific example, the monoclonal antibody according to the methodof the invention is Rituximab. In certain embodiments, the carbohydratemoiety according to the method the invention is selected from the groupconsisting of Sia₂(α2-6)Gal₂GlcNAc₂Man₃GlcNAc,Sia₂(α2-6)Gal₂GlcNAc₃Man₃GlcNAc, Sia₂(α2-3)Gal₂GlcNAc₂Man₃GlcNAc,Sia₂(α2-3)Gal₂GlcNAc₃Man₃GlcNAc, Sia₂(α2-3/α2-6)Gal₂GlcNAc₂Man₃GlcNAc,Sia₂(α2-6/α2-3)Gal₂GlcNAc₂Man₃GlcNAc,Sia₂(α2-3/α2-6)Gal₂GlcNAc₃Man₃GlcNAc,Sia₂(α2-6/α2-3)Gal₂GlcNAc₃Man₃GlcNAc, Sia(α2-6)Gal₂GlcNAc₂Man₃GlcNAc,Sia(α2-3)Gal₂GlcNAc₂Man₃GlcNAc, Sia(α2-6)Gal₂GlcNAc₃Man₃GlcNAc,Sia(α2-3)Gal₂GlcNAc₃Man₃GlcNAc, Sia(α2-6)GalGlcNAc₂Man₃GlcNAc,Sia(α2-3)GalGlcNAc₂Man₃GlcNAc, Sia(α2-6)GalGlcNAc₃Man₃GlcNAc,Sia(α2-3)GalGlcNAc₃Man₃GlcNAc, Gal₂GlcNAc₂Man₃GlcNAc andGal₂GlcNAc₃Man₃GlcNAc.

In some embodiments, the carbohydrate moiety is a sugar oxazoline.

Step (b) in the method of the invention may lead to sugar chainextension. One method for sugar chain extension is through anenzyme-catalyzed glycosylation reaction. It is well known in the artthat glycosylation using a sugar oxazoline as the sugar donor among theenzyme-catalyzed glycosylation reactions is useful for synthesizingoligosaccharides because the glycosylation reaction is an additionreaction and advances without any accompanying elimination of acid,water, or the like. (Fujita, et al., Biochim. Biophys. Acta 2001, 1528,9-14)

Suitable conditions in step (b) include incubation of the reactionmixture for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60minutes, 70 minutes, 80 minutes, 90 minutes or 100 minutes, preferablyless than 60 minutes. Incubation preferably takes place at roomtemperature, more preferably at approximately 20° C., 25° C., 30° C.,35° C., 40° C. or 45° C., and most preferably at approximately 37° C.

As used herein, the terms “fucose” and “L-fucose” are usedinterchangeably.

As used herein, the terms “core fucose” and “core fucose residue” areused interchangeably and refer to a fucose in α1,3-position orα1,6-position linked to the asparagine-bound N-acetylglucosamine.

As used herein, the term “α-(1,2) Fucosidase” refers to anexoglycosidase that specifically catalyzes the hydrolysis of α-(1,2)linked L-fucose residues from oligosaccharides.

As used herein, the term “α-(1,4) Fucosidase” refers to anexoglycosidase that specifically catalyzes the hydrolysis of α-(1,4)linked L-fucose residues from oligosaccharides.

As used herein, the term “glycan” refers to a polysaccharide,oligosaccharide or monosaccharide. Glycans can be monomers or polymersof sugar residues and can be linear or branched. A glycan may includenatural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose,xylose, etc.) and/or modified sugars (e.g., 2′-fluororibose,2′-deoxyribose, phosphomannose, 6′ sulfo N-acetylglucosamine, etc).

As used herein, the terms “N-glycan”, “N-linked glycan”, “N-linkedglycosylation”, “Fc glycan” and “Fc glycosylation” are usedinterchangeably and refer to an N-linked oligosaccharide attached by anN-acetylglucosamine (GlcNAc) linked to the amide nitrogen of anasparagine residue in a Fc-containing polypeptide. The term“Fc-containing polypeptide” refers to a polypeptide, such as anantibody, which comprises an Fc region.

As used herein, the term “glycosylation pattern” and “glycosylationprofile” are used interchangeably and refer to the characteristic“fingerprint” of the N-glycan species that have been released from aglycoprotein or antibody, either enzymatically or chemically, and thenanalyzed for their carbohydrate structure, for example, using LC-HPLC,or MALDI-TOF MS, and the like. See, for example, the review in CurrentAnalytical Chemistry, Vol. 1, No. 1 (2005), pp. 28-57; hereinincorporated by reference in its entirety.

As used herein, the term “glycoengineered Fc” when used herein refers toN-glycan on the Fc region has been altered or engineered eitherenzymatically or chemically. The term “Fc glycoengineering” as usedherein refers to the enzymatic or chemical process used to make theglycoengineered Fc.

The terms “homogeneous”, “uniform”, “uniformly” and “homogeneity” in thecontext of a glycosylation profile of Fc region are used interchangeablyand are intended to mean a single glycosylation pattern represented byone desired N-glycan species, with no trace amount of precursorN-glycan.

As used herein, the terms “IgG”, “IgG molecule”, “monoclonal antibody”,“immunoglobulin”, and “immunoglobulin molecule” are usedinterchangeably. As used herein, “molecule” can also include antigenbinding fragments.

As used herein, the term “glycoconjugate”, as used herein, encompassesall molecules in which at least one sugar moiety is covalently linked toat least one other moiety. The term specifically encompasses allbiomolecules with covalently attached sugar moieties, including forexample N-linked glycoproteins, O-linked glycoproteins, glycolipids,proteoglycans, etc.

As used herein, the term “glycolipid” refers to a lipid that containsone or more covalently linked sugar moieties (i.e., glycans). The sugarmoiety(ies) may be in the form of monosaccharides, disaccharides,oligosaccharides, and/or polysaccharides. The sugar moiety(ies) maycomprise a single unbranched chain of sugar residues or may be comprisedof one or more branched chains. In certain embodiments, sugar moietiesmay include sulfate and/or phosphate groups. In certain embodiments,glycoproteins contain O-linked sugar moieties; in certain embodiments,glycoproteins contain N-linked sugar moieties.

As used herein, the term “glycoprotein” refers to amino acid sequencesthat include one or more oligosaccharide chains (e.g., glycans)covalently attached thereto. Exemplary amino acid sequences includepeptides, polypeptides and proteins. Exemplary glycoproteins includeglycosylated antibodies and antibody-like molecules (e.g., Fc fusionproteins). Exemplary antibodies include monoclonal antibodies and/orfragments thereof, polyclonal antibodies and/or fragments thereof, andFc domain containing fusion proteins (e.g., fusion proteins containingthe Fc region of IgG1, or a glycosylated portion thereof).

As used herein, the term “N-glycan” refers to a polymer of sugars thathas been released from a glycoconjugate but was formerly linked to theglycoconjugate via a nitrogen linkage (see definition of N-linked glycanbelow).

As used herein, the term “O-glycan” refers to a polymer of sugars thathas been released from a glycoconjugate but was formerly linked to theglycoconjugate via an oxygen linkage (see definition of O-linked glycanbelow).

As used herein, a functional variant of a wild-type enzyme possesses thesame enzymatic activity as the wild-type counterpart and typicallyshares a high amino acid sequence homology, e.g., at least about 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of thewild-type counterpart. The “percent identity” of two amino acidsequences is determined using the algorithm of Karlin and Altschul Proc.Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin andAltschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithmis incorporated into the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to the protein molecules ofinterest. Where gaps exist between two sequences, Gapped BLAST can beutilized as described in Altschul et al., Nucleic Acids Res.25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs,the default parameters of the respective programs (e.g., XBLAST andNBLAST) can be used. A functional variant can have various mutations,including addition, deletion, or substitution of one or more amino acidresidues. Such a variant often contain mutations in regions that are notessential to the enzymatic activity of the wild-type enzyme and maycontain no mutations in functional domains or contain only conservativeamino acid substitutions. The skilled artisan will realize thatconservative amino acid substitutions may be made in lipoic acid ligasemutants to provide functionally equivalent variants, i.e., the variantsretain the functional capabilities of the particular lipoic acid ligasemutant.

As used herein, a “conservative amino acid substitution” refers to anamino acid substitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g. MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D. Any of the enzymes involved in thedeglycosylation system can be prepared via routine technology. In oneexample, the enzyme is isolated form a natural source. In otherexamples, the enzyme is prepared by routine recombinant technology. Whennecessary, the coding sequence of a target enzyme can be subjected tocoden optimization based on the host cell used for producing the enzyme.For example, when E. coli cells are used as the host for producing anenzyme via recombinant technology, the gene encoding that enzyme can bemodified such that it contains codons commonly used in E. coli. Thedetails of one or more embodiments of the invention are set forth in thedescription below. Other features or advantages of the present inventionwill be apparent from the following drawings and detailed description ofseveral embodiments, and also from the appending claims.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1 Protein Expression Constructs

The α-fucosidases were amplified by PCR from Bacteroides fragilis NCTC9343 genomic DNA (ATCC 25285) and Bacteroides Thetaiotaomicron VPI-5482(ATCC 29148), respectively, and cloned into pET47b+ (EMD Biosciences,San Diego, Calif.) with N-terminal poly-histine with internal AcTEVprotease cutting site. Other enzymes used in the study such as Endo F1(GenBank: AAA24922.1), Endo F2 (GenBank: AAA24923.1), Endo F3 (GenBank:AAA24924.1), Endo H (GenBank: AAA26738.1) and PNGase F (Genbank:GenBank: J05449.1) were codon optimized for E. coli, and cloned intopET28a with MBP fusion in N-terminus, respectively. All sequences of theclones were first confirmed by Applied Biosystems 3730 DNA Analyzer.

Primers used for protein expression constructs in E. coli are listed inthe table below.

Restric- Gene source tion from genome SEQ ID enzyme or cDNA NOPrimer^(a) Sequence (5′→3′) site pool SEQ ID BfFucH- TTCAGGGA GCGATCGCTCAGCAAAAGTATCAACCGACA^(b) AsiSI Bacteroides NO: 3 F fragilis SEQ IDBfFucH- GTCATTAC GTTTAAAC TTAGTCAATTGTAAGTTCTACCA PmeI (BfFucH, e.g.NO: 4 R GenBank accession no. YP_212855.1) SEQ ID BtFucH- TTCAGGGAGCGATCGC TCAGTCTTCTTACCAGCCTGGT AsiSI Bacteroides NO: 5 F thetaiotao-SEQ ID BtFucH- GTCATTAC GTTTAAAC TTAGTCAATTGTAAGTTCTACAAC PmeI micronNO: 6 R (BtFucH, e.g. GenBank accession no. AAO76949.1) SEQ ID EndoF1-TTCAGGGAG CGATCGC TGCGGTTACCGGTACCACCA AsiSI Elizabeth- NO: 7 Fkingia miricola SEQ ID EndoF1- GTCATTAC GTTTAAAC TTACCAGTCTTTAGAGTACGGGGPmeI (e.g. Genbank NO: 8 R accession no. AAA24922.1) SEQ ID EndoF2-TTTCAGGGA GCGATCGC TGCGGTTAACCTGTCTAACCT AsiSI Elizabeth- NO: 9 Fkingia miricola SEQ ID EndoF2- GTCATTAC GTTTAAACTTACGGGTTCATGATTTTGATCAG PmeI (e.g. GenBank NO: 10 R accession no.AAA24923.1) SEQ ID EndoF3- TTCAGGGA GCGATCGC TGCGACCGCGCTGGCGGGTT AsiSIElizabeth- NO: 11 F kingia miricola SEQ ID EndoF3- GTCATTAC GTTTAAACTTAGTTTTTAACCGCGTCACGAAC PmeI (e.g. GenBank NO: 12 R accession no.AAA24924.1) SEQ ID EndoH- TTCAGGGA GCGATCGC TGCGCCGGCGCCGGTTAAACA AsiSIStreptomyces NO: 13 F plicatus (e.g. SEQ ID EndoH- GTCATTAC GTTTAAACTTACGGGGTACGAACCGCTTCAG PmeI GenBank NO: 14 R accession no. AAA26738.1)SEQ ID endoS-F TTCAGGGA GCGATCGC TACCCACCATGATTCACTCAAT AsiSIStreptococcus NO: 15 pyogenes (e.g. SEQ ID endoS-R GTCATTAC GTTTAAACTTATTTTTTTAGCAGCTGCCTTTTC PmeI GenBank NO: 16 accession no. AAK34539.1)SEQ ID PNGase TTCAGGGA GCGATCGC TGCGCCGGCGGACAACACCGT AsiSI Chryseobac-NO: 17 F-F terium SEQ ID PNGase GTCATTAC GTTTAAACTTAGTTGGTAACAACCGGCGCAGA PmeI meningosep- NO: 18 F-R ticum (e.g. GenBankaccession no. J05449.1) ^(a)a pair of primers for forward (F) andreversed (R) PCR reactions to amplify the coding sequence of each gene.^(b)Underline with bold means the site of restriction enzymerecognition. ^(c)Codon optimization for E. coli. See, e.g., Puigbò etal., Nucleic Acids Research (2007) 35(S2):W126-W130.Protein Expression and Purification

Protein expression constructs were transformed into BL21(DE3) (EMDBiosciences, San Diego, Calif.) for protein expression using 0.2 mMisopropyl β-D-thiogalactopyranoside (IPTG) in 16° C. for 24 hours. Cellswere disrupted by microfluidizer and then centrifuged. Supernatants werecollected and loaded onto Ni-NTA agarose column (QIAGEN GmbH, Hilden,Germany) and washed with ten folds of washing buffer (sodium phosphatebuffer (pH 7.0), 300 mM sodium chloride, and 10 mM imidazole). Elutionwas employed by two folds of elution buffer (sodium phosphate buffer (pH7.0), 300 mM sodium chloride, and 250 mM imidazole), followed by bufferexchanging into reaction buffer by Amicon Ultra-15 10K (EMD MilliporeChemicals, Billerica, Mass.). Protein purity was examined by SDS-PAGEand quantitative protein concentration was measured by Qubit® ProteinAssay Kits (Invitrogen, Carlsbad, Calif.). The recombinant fucosidasewith his-tag followed by Ni-NTA column purification resulted in a yieldof 60 mg/L with greater than 95% purity. Protein concentration wasdetermined according to the method of Brandford (Protein Assay; Bio-Rad,Hercules, Calif., USA) with bovine serum albumin as standards. Thepurity and molecular mass of the enzyme was examined by SDS-PAGE.

The purified fucosidase from Bacteroides fragilis exhibited a molecularmass of about 50 kDa in sodium dodecylsulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) which is close to the theoretical molecularweight of 47.3 kDa.

Example 2 Enzymatic Assays

Characteristics of Enzymes

Unlike fucosidases from mammalian or bacterias, which have optimum pH inthe acid condition (pH 4.0-6.0), BfFucH preformed well at mild condition(pH 7.0-7.5). In addition, BfFucH is not affected by certain divalentmetal ions, and exogenous addition of metal ions did not influence theactivity. However, Ni²⁺ can dramatically reduce the enzymatic activityby 60%. Also, Zn²⁺ and Cu²⁺ can completely abolish the enzymaticactivity. The chelator EDTA showed no effect on the enzymatic activity,indicating the metal ions do not participate in the catalytic reaction.The enzyme is functionally active and stable at room temperate and at 4°C.

Enzyme Activity on N-Linked Glycas

The fucosidases described herein can be used to determine the fucoseposition in N-glycan. The BfFucH hydrolysis activity on N-glycans withvarious fucoses attached at different positions was evaluated. Twosynthetic glycopeptides, 0800F and 0823F, were prepared. Bothglycopeptides have the fucoses bound to outer GlcNAc and the innermostGlcNAc at the glycosylation site, respectively.

Enzymatic assays revealed that the fucose could be released only fromthe outer GlcNAc in the sample 0800F, but not in the glycopeptide 0823Fwhere the fucose is bound to the innermost GlcNAc. This result indicatedthat the steric hindrance of the G0 structure in N-glycan may shield andprotect the fucose from fucosidase hydrolysis. In contrast, if 0823F wastreated with BfFucH and endo-β-N-acetylglucosaminidase (endo M)simultaneously in a one-pot reaction, the core fucose could easily beremoved. This result indicated that the α-fucosidase can be used todistinguish the position of fucose bound to a glycan.

Enzyme Activity on Oligosaccharides

The lipopolysaccharide (LPS) of serotypes O86, O128, and O111 of E. colistrains contains various monosaccharides, e.g., Gal, GalNAc, and fucose.By the formaldehyde dehygrogenase (FDH) coupled assay, we confirmed thatBfFucH can liberate L-fucose from LPS of E. coli O128:B12 strain in adose-dependent manner. We also tested the enzymatic activity of theenzyme on various substrates including 2′-fucosyllactose (2′FL),3′-fucosyllactose (3′FL), lacto-N-fucopentaose I (LNPT I), Globo H,Lewis a (Le^(a)), Lewis x (Le^(x)), Lewis b (Le^(b)), Lewis y (Le^(y)),Sialyl Lewis a (SLe^(a)), Sialyl Lewis x (SLe^(x)), and pNP(para-Nitrophenol)-α-L-fucoside. Results showed the α-fucosidase areable to hydrolyze all of the substrates.

Example 3 Core Defucosylation of Glycoproteins

Aleuria aurantia possesses a fucose-specific lectin (AAL) that is widelyused as a specific probe for fucose. AAL recognizes and bindsspecifically to fucose and terminal fucose residues on complex oligosaccharides and glycoconjugates. AAL can be used to determine the coredefucosylation. Endoglycosidase is useful for trimming off the variableportions of an oligosaccharide in the N-glycan. After the treatment of acocktail of endoglycosidases (Endo F1, Endo F2, Endo F3 and Endo H), theantibody (Humira or Rituxan) showed a high AAL-blotting signal,indicating the presence of core fucose in the antibody. However, afterthe treatment of a combination of a cocktail of endoglycosidases (EndoF1, Endo F2, Endo F3 and Endo H) and BfFucH, the antibody (Humira orRituxan) lost the AAL-blotting signal due to the hydrolysis of corefucose. These results demonstrated the BfFucH is active for coredefucosylation.

Materials and Methods

Unless otherwise noted, all compounds and reagents were purchased fromSigma-Aldrich or Merck. Anti-tumor necrosis factor-alpha (TNFα)antibody, Adalimumab (Humira®), was purchased from (North Chicago,Ill.). Anti-human CD20 mouse/human chimeric IgG1 rituximab (Rituxan®)was purchased from Genentech, Inc. (South San Francisco, Calif.)/IDECPharmaceutical (San Diego, Calif.). TNF receptor-Fc fusion proteinEtanercept (Enbrel®) was purchased from Wyeth Pharmaceuticals(Hampshire, UK). Epoetin beta (Recormon®) was purchased from Hoffmann-LaRoche Ltd (Basel, Switzerland). Interferon β1a (Rebif®) was purchasedfrom EMD Serono, Inc. (Boston, Mass.).

Para-Nitrophenyl α- or β-monosaccharide, Lewis sugars, blood type sugarsand human milk oligosaccharides were purchased from Carbosynth Limited.(Berkshire, UK). Primary antibody against IgG Fc region, Recormon®, andRebif® were purchased from Chemicon (EMD Millipore Chemicals, Billerica,Mass.). Biotinylated Aleuria Aurantia Lectin (AAL) and HRP-ConjugatedStreptavidin were purchased from Vector Laboratory (Burlingame, Calif.).Chemiluminescence on protein blots was visualized and quantified usingthe ImageQuant LAS 4000 biomolecular imager system.

BfFucH Activity Analytical Methods

Enzyme activity was measured at 25° C. in 50 mM sodium phosphate buffer,pH 7.0, using pNP-α-L-Fuc (p-nitrophenyl-α-L-Fuc) as a substrate, asstandard assay condition. One unit of α-L-fucosidase activity wasdefined as the formation of μmol of pNP and Fuc from the pNP-α-L-Fuc perminute in 50 mM sodium phosphate buffer, pH 7.0, at 25° C. Values forMichaelis constants (Km), Turnover Number (Kcat) and Vmax werecalculated for pNP-α-L-Fuc from the Michaelis-Menten equation bynon-linear regression analysis by GraphPad Prism v5 software (La Jolla,Calif.).

Activity Measurement of Optimum pH of BfFucH.

The optimum pH for fucosidase activity was determined in the standardenzyme assay mentioned above in the pH range 4.0-10.0, including sodiumacetate, MES, MOPS, HEPES, Tris-HCl, CHES buffer. All reactions wereperformed in triplicate for statistical evaluation.

Activity Measurement of Optimum Divalent Metal Ion of BfFucH

The assay for metal requirement was performed in standard assaycondition. Enzymes were mixed with metal ion (Mg²⁺, Mn²⁺, Ca²⁺, Zn²⁺,Co²⁺, or Fe²⁺, Cu²⁺) in a final concentration of 5 mM, in the presenceand absence of EDTA. All reactions were performed in triplicate forstatistical evaluation.

Activity Measurement of Optimum Temperature of BfFucH

The effect of temperature on the activity of enzymes were determined byincubating sufficient amount of purified fucosidase with pNP-α-L-Fuc insodium phosphate buffer (pH 7.0). In order to keep the assay consist,all components were mixed well and preheated at assay temperature for 10min, and the reaction was started by adding the enzyme and recorded bymultimode plate readers (SpectraMax M5, Molecular Devices) in constanttemperature. The temperature ranged from 4 to 80° C. All reactions wereperformed in triplicate for statistical evaluation.

Fucose Dehydrogenase-Based (FDH) Assay

The fucose dehydrogenase-based assay was slightly modified from previousreports. Unlike other fucose dehydrogenases from Pseudomonas sp sold bySigma-Aldrich, which are active only react with NADP+, the recombinantform of FDH from Mesorhizobium loti are functional with only NAD+. TheNADH formed was measured by NADPH fluorescence at around 450 nm whenexcited with 340 nm by multimode plate readers (SpectraMax M5, MolecularDevices) at 25° C. By using this method, fucosyl-conjugates in variousoligosaccharides such as Lewis sugar and human milk oligosaccharides(HMOs) were quantitated within 5 min.

Generation of Mono-GlcNAc or GlcNAc-(Fuc α-1,6) of Immunoglobulin G,Fc-Fusion Protein, EPO, Interferon (IFNβ1a) and Influenza Hemagglutinin(HA)

All the glycoproteins were buffer exchanging by reaction buffer 50 mMsodium phosphate buffer (pH 7.0). First the endoglycosidases cocktailsolution, including EndoF1, EndoF2, EndoF3, EndoH and EndoS (1 mg/mL),were added in order to remove all the N-glycan chain except the GlcNAcbound to Asn of glycoproteins followed by the suitable quantities offucosidase. Incubate at 37° C. for 48 hours in order to completelyremove the core-fucose bound to GlcNAc of glycoproteins.

We claim:
 1. A composition comprising an α-fucosidase, wherein thecomposition comprises: (a) an α-fucosidase polypeptide having thesequence of SEQ ID NO: 1; (b) at least one glycosidase selected from thegroup consisting of Endo-beta-N-acetylglucosaminidases (NAG), EndoA,EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, and variants thereof; and(c) a glycoprotein comprising an N-linked glycan, wherein theglycoprotein is selected from the group consisting of an immunoglobulinG, an Fc-fusion protein, erythropoietin, interferon and influenzahemagglutinin.
 2. The composition of claim 1, wherein the α-fucosidaseis an isolated polypeptide.
 3. The composition of claim 1, wherein theα-fucosidase is a recombinant Bacteroides α-L-fucosidase.
 4. Thecomposition of claim 1, wherein the α-fucosidase can hydrolyze α-(1,2),α-(1,3), α-(1,4), and α-(1,6)-linked fucoses present in N- and/orO-linked glycans in a glycoconjugate.
 5. The composition of claim 1,wherein the α-fucosidase has a pH optimum from about pH 7.0 to about pH7.5.
 6. The composition of claim 1, wherein the immunoglobulin Gcomprises an anti-CD20 antibody or an anti-TNFα antibody.
 7. Thecomposition of claim 6, wherein the anti-CD20 antibody is a chimericIgG1.
 8. The composition of claim 7, wherein the anti-CD20 antibody isrituximab.
 9. The composition of claim 6, wherein the anti-TNFα antibodyis adalimumab.
 10. A composition comprising an α-fucosidase wherein thecomposition comprises: (a) an α-fucosidase polypeptide having the aminoacid sequence of SEQ ID NO: 1; (b) at least one glycosidase selectedfrom the group consisting of Endo-beta-N-acetylglucosaminidases (NAG),EndoA, EndoF1, EndoF2, EndoF3, EndoH, EndoM, EndoS, and variantsthereof; and (c) a glycoprotein selected from the group consisting of animmunoglobulin G, a Fc-fusion protein, erythropoietin, interferon andinfluenza hemagglutinin.
 11. The composition of claim 10, whereinα-fucosidase is a recombinant Bacteroides α-L-fucosidase.
 12. Thecomposition of claim 10, wherein the α-fucosidase can hydrolyze α-(1,2),α-(1,3), α-(1,4), and α-(1,6)-linked fucoses present in N- and/orO-linked glycans in a glycoconjugate.
 13. A composition comprising anα-fucosidase wherein the composition comprises: (a) an α-fucosidasepolypeptide having the sequence of SEQ ID NO: 1; (b) at least oneglycosidase selected from the group consisting ofEndo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2, EndoF3,EndoH, EndoM, EndoS, and variants thereof; and (c) a glycoproteincomprising an N-linked glycan, wherein the glycoprotein is selected fromthe group consisting of an immunoglobulin G, an Fc-fusion protein,erythropoietin, interferon and influenza hemagglutinin; and wherein thepH of the composition is between about 7.0 to about 8.0.
 14. Acomposition comprising an α-fucosidase wherein the compositioncomprises: (a) an α-fucosidase polypeptide having the sequence of SEQ IDNO: 1; (b) at least one glycosidase selected from the group consistingof Endo-beta-N-acetylglucosaminidases (NAG), EndoA, EndoF1, EndoF2,EndoF3, EndoH, EndoM, EndoS, and variants thereof; and (c) aglycoprotein comprising an N-linked glycan, wherein the glycoprotein isselected from the group consisting of an immunoglobulin G, an Fc-fusionprotein, erythropoietin, interferon and influenza hemagglutinin; andwherein the composition excludes a divalent cation selected from thegroup consisting of Zn²⁺, Cu²⁺ and Ni²⁺.
 15. A composition comprising anα-fucosidase, wherein the composition comprises: (a) an α-fucosidasepolypeptide having the sequence of SEQ ID NO: 1; (b) at least oneglycosidase selected from the group consisting of EndoF1, EndoF2,EndoF3, EndoH, EndoS, and variants thereof; and (c) a glycoproteincomprising an N-linked glycan, wherein the glycoprotein is selected fromthe group consisting of an immunoglobulin G, an Fc-fusion protein,erythropoietin, interferon and influenza hemagglutinin.
 16. Acomposition comprising an α-fucosidase, wherein the compositioncomprises: (a) an α-fucosidase polypeptide having the sequence of SEQ IDNO: 1; and (b) (i) Endo S and optionally at least one glycosidaseselected from the group consisting of EndoF1, EndoF2, EndoF3, EndoH,EndoS, and variants thereof; or (ii) a glycosidase cocktail comprisingEndo F1, Endo F2, Endo F3, and Endo H; and (c) an immunoglobulin G or anFc-fusion protein comprising an N-linked glycan.
 17. A composition madeby a process comprising: contacting a glycoprotein with an α-fucosidaseto remove one or more fucoses in the glycoprotein, wherein thecomposition comprises an α-fucosidase polypeptide having the sequence ofSEQ ID NO: 1 and a glycosidase cocktail comprising EndoF1, EndoF2,EndoF3, EndoH and EndoM, wherein the glycoprotein comprises an N-linkedglycan and is selected from the group consisting of an immunoglobulin G,an Fc-fusion protein, erythropoietin, interferon and influenzahemagglutinin.